CDKI pathway inhibitors and uses thereof

ABSTRACT

The invention relates to compounds for inhibiting the Cyclin-Dependent Kinase Inhibitor (CDKI) pathway. More particularly, the invention relates to compounds for inhibiting the CDKI pathway for studies of and intervention in senescence-related and other CDKI-related diseases.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the inhibition of the Cyclin-Dependent KinaseInhibitor (CDKI) pathway. More particularly, the invention relates tocompounds and methods for inhibiting the CDKI pathway for studies of andintervention in senescence-related diseases, including degenerativediseases of the central nervous system, including Alzheimer's Diseaseand other dementias, as well as for studies of and intervention incancer and viral diseases.

2. Summary of the Related Art

Cell senescence, originally defined as a series of cellular changesassociated with aging, is now viewed more broadly as a signaltransduction program leading to irreversible cell cycle arrest,accompanied by a distinct set of changes in the cellular phenotype (Seee.g. Campisi, Cell 120: 513-522 (2005); Shay and Roninson, Oncogene 23:2919-2933 (2004)). Senescence can be triggered by many differentmechanisms including the shortening of telomeres (replicativesenescence) or by other endogenous and exogenous acute and chronicstress signals, including major environmental factors, such as UV andcigarette smoke. The latter forms of telomere-independent senescence arevariably referred to as accelerated senescence, STASIS (Stress orAberrant Signaling Induced Senescence), or SIPS (Stress-InducedPremature Senescence). Regardless of the mode of induction, senescentcells develop the same general phenotype, characterized not only bypermanent growth arrest but also by enlarged and flattened morphology,increased granularity, high lysosomal mass, and expression ofsenescence-associated endogenous β-galactosidase activity (SA-β-gal).

Dimri et al., Proc. Natl. Acad. Sci. USA 92: 9363-9367 (1995) teachesthat in the human body, the phenotype of cell senescence has beendetected in correlation with aging. Castro et al., Prostate 55: 30-38(2003); Michaloglou et al., Nature 436: 720-724 (2005); and Collado etal., Nature 436: 642 (2005) teach that the phenotype of cell senescencehas also been detected in pathological situations, including variouspre-malignant conditions. te Poele et al., Cancer Res. 62: 1876-1883(2002); and Roberson et al., Cancer Res. 65: 2795-2803 (2005) teach itsdetection in many tumors treated with chemotherapy.

In most systems of senescence that have been characterized at themolecular level, cell cycle arrest is triggered by the activation ofp53, which in its turn induces a broad-specificity cyclin-dependentkinase inhibitor (CDKI) p21^(Waf1/Cip1/Sdi1). p21 induction causes cellcycle arrest at the onset of senescence, but p53 and p21 levels decreaseat a later stage. Shay and Roninson, Oncogene 23: 2919-2933 (2004) teachthat this decrease is accompanied, however, by a stable increase inanother CDKI protein, p16^(Ink4A), which is believed to be primarilyresponsible for the maintenance of cell cycle arrest in senescent normalcells.

CDKI proteins act as negative regulators of the cell cycle and aretherefore generally known as tumor suppressors. The induction of CDKIproteins, in particular p21, also occurs in tumor cells in the contextof cancer therapy, in response to cellular damage by different classesof cancer chemotherapeutic drugs and ionizing radiation. Cell cyclearrest by CDKIs mediates the cytostatic and senescence-inducing activityof anticancer agents, one of the major components of their therapeuticeffect (Roninson, Cancer Res., 11, 2705-2715). Agents that would enhancethe ability of CDKI proteins to induce cell cycle arrest will thereforebe useful for the chemoprevention of cancer and for increasing thetherapeutic efficacy of conventional anticancer agents.

Although senescent cells do not divide, they remain fully viable,metabolically and synthetically active. It has now been recognized thatsenescent cells secrete a variety of factors that have a major effect ontheir environment. Campisi, supra teaches that secretory activities ofsenescent cells have been linked to carcinogenesis, skin aging, and avariety of age-related diseases. A series of studies have implicated p21and other CDKI proteins in disease-promoting activities of senescentcells. This insight came principally from the analysis by Chang et al.,Proc. Natl. Acad. Sci. USA 97: 4291-4296 (2000) of the transcriptionaleffects of p21, expressed in a fibroblastoid cell line from an induciblepromoter. This analysis showed that p21 produces significant changes inthe expression of multiple genes. Many genes are strongly and rapidlyinhibited by p21, and most of these are involved in cell proliferation.Zhu et al., Cell Cycle 1: 50-58 (2002) teaches that inhibition of cellcycle progression genes by p21 is mediated by negative cis-regulatoryelements in the promoters of these genes, such as CDE/CHR. The samegenes are downregulated in tumor cells that undergo senescence afterchemotherapeutic treatment, but Chang et al., Proc. Natl. Acad. Sci. USA99: 389-394 (2002) teaches that p21 knockout prevents the inhibition ofthese genes in drug-treated cells. Hence, p21 is responsible for theinhibition of multiple cell cycle progression genes in response to DNAdamage.

Chang et al., 2000, supra teaches that another general effect of p21induction is upregulation of genes, many of which encode transmembraneproteins, secreted proteins and extracellular matrix (ECM) components.This effect of p21 is relatively slow, occurring subsequently to growtharrest and concurrently with the development of the morphologicalfeatures of senescence. These genes are induced by DNA damage but p21knockout decreases their induction (Chang et al., 2002, supra). Thisdecrease is only partial, which can be explained by recent findings bythat the majority of p21-inducible genes are also induced in response toother CDKI, p16 and p27 (see WO 03/073062). Gregory et al., Cell Cycle1: 343-350 (2002); and Poole et al., Cell Cycle 3: 931-940 (2004) teachthat gene upregulation by CDKI has been reproduced using promoterconstructs of many different CDKI-inducible genes, indicating that itoccurs at the level of transcription. (Perkins et al., Science 275:523-527 (1997); Gregory et al., supra; and Poole et al., supra teachthat induction of transcription by p21 is mediated in part bytranscription factor NFκB and transcription cofactors of p300/CBPfamily, but other intermediates in the signal transduction pathway thatleads to the activation of transcription in response to CDKI—the CDKIpathway—remain presently unknown (FIG. 1).

Medical significance of the induction of transcription by CDKI has beenindicated by the known functions of CDKI-inducible genes (Chang et al.,2000, supra). Many CDKI-upregulated genes are associated with cellsenescence and organism aging, including a group of genes implicated inage-related diseases and lifespan restriction. One of these genes isp66^(Shc), a mediator of oxidative stress, the knockout of which expandsthe lifespan of mice by about 30% (Migliaccio et al., supra). ManyCDKI-induced genes play a role in age-related diseases, most notablyAlzheimer's disease and amyloidosis. Thus, CDKI induce many humanamyloid proteins, including Alzheimer's amyloid β precursor protein(βAPP) and serum amyloid A, implicated in amyloidosis, atherosclerosisand arthritis. CDKI also upregulate tissue transglutaminase thatcross-links amyloid peptides leading to plaque formation in bothAlzheimer's disease and amyloidosis. Some of CDKI-inducible genes areconnective tissue growth factor and galectin-3 involved inatherosclerosis, as well as cathepsin B, fibronectin and plasminogenactivator inhibitor 1, associated with arthritis. Murphy et al., J.Biol. Chem. 274: 5830-5834 (1999) teaches that several CDKI-inducibleproteins are also implicated in an in vitro model of nephropathy.Remarkably, p21-null mice were found to be resistant to experimentalinduction of atherosclerosis (Merched and Chan, Circulation 110:3830-3841 (2004)) and chronic renal disease (Al Douahji et al., KidneyInt. 56: 1691-1699 (1999); Megyesi et al., Proc. Natl. Acad. Sci. USA96: 10830-10835 (1999).

In addition to their effect on cellular genes, CDKI stimulate thepromoters of many human viruses, such as HIV-1, cytomegalovirus,adenovirus and SV40. Since many viruses induce p21 expression ininfected cells, this effect suggests that promoter stimulation by CDKImay promote viral infections (Poole et al., supra).

Strong associations for CDKI-inducible genes have also been found incancer. In particular, p21 expression activates the genes for manygrowth factors, inhibitors of apoptosis, angiogenic factors, andinvasion-promoting proteases. In accordance with these changes in geneexpression, Chang et al., 2000, supra teaches that p21-arrested cellsshow paracrine mitogenic and anti-apoptotic activities in cocultureassays. Krtolica et al., Proc. Natl. Acad. Sci. USA 98: 12072-12077(2001) teaches that paracrine tumor-promoting activities weredemonstrated both in vitro and in vivo in CDKI-expressing normalsenescent fibroblasts, which express p21 and p16. Importantly, senescentfibroblasts possess the characteristic pro-carcinogenic activity thathas long been identified with tumor-associated stromal fibroblasts.Furthermore, all the experimental treatments shown to endow fibroblastswith tumor-promoting paracrine activities also induce CDKI, suggestingthat the CDKI pathway could be the key mediator of pro-carcinogenicactivity of stromal fibroblasts (Roninson, Cancer Lett. 179: 1-14(2002)).

CDKI expression mediates cell cycle arrest not only in the program ofsenescence but also in numerous other situations, such as transientcheckpoint arrest in response to different forms of damage, contactinhibition, and terminal differentiation. Hence, the CDKI pathway, whichleads to the activation of multiple disease-promoting genes, isactivated not only in cell senescence but also in many otherphysiological situations. As a result, CDKI-responsive gene products areexpected to accumulate over the lifetime, contributing to thedevelopment of Alzheimer's disease, amyloidosis, atherosclerosis,arthritis, renal disease, viral diseases, including HIV/AIDS and cancer.

The effects of CDKIs are usually considered in light of their inhibitionof cyclin-dependent kinases (CDKs), a family of serine/threonine kinasescomprising 21 members in the human genome, which act in a complex withregulatory cyclin proteins. The best-known CDKs (CDK1, CDK2, CDK4, CDK6)are required for transitions between different phases of the cell cycle,but many other CDKs function as regulators of transcription or RNAprocessing rather than the cell cycle (Malumbres et al., 2009). Amongthe latter, of special relevance to the instant invention are CDK8 and aclosely related CDK19 (80% overall identity, 98% identity in the ATPpocket). These CDKs, coupled with Cyclin C, are alternative componentsof a regulatory module of the Mediator complex that connectstranscriptional regulators with RNA polymerase II to initiatetranscription (Sato et al., 2004). CDK19 has been also called CDC2L6and, confusingly, CDK11, but the name CDK11 is more often applied to twoother proteins, presently known as CDK11A and CDK11B (Malumbres et al.,2009). CDK8 has been the subject of great attention in recent years andwas identified as playing an important role in cancer (reviewed inFirestein and Hahn, 2009). CDK8 knockdown and knockout studies showedthat it is not needed for cell growth but is required for earlyembryonic development (Westerling et al., 2007). CDK8 has beenassociated with processes involved in senescence and damage response: itregulates Smad transcriptional activation and turnover in BMP and TGF-βpathways (Alarcon et al., 2009) and acts as a stimulus-specific positivecoregulator of p53 target genes (Donner et al., 2007). CDK8 has beenidentified as an oncogene amplified in ˜50% of colon cancers, acting asa positive regulator of β-catenin, a transcription factor that plays acentral role in colon carcinogenesis (Firestein et al., 2008; Morris etal., 2008). CDK8 has not yet been implicated in human diseases otherthan cancer.

In contrast to CDK8, little is known about CDK19, which substitutes forCDK8 in the corresponding Mediator modules but may have an oppositeeffect on the regulation of transcription. In particular, the study ofTsutsui et al. (2008), which refers to CDK19 as CDK11, reports thatCDK19 acts as a negative regulator of viral activator VP16-dependenttranscription, in contrast to CDK8 that acts as a positive regulator inthis system. Pohlner and Von der Kammer (US patent publication2009/00047274 A1) report that CDK19 (called there CDC2L6) is upregulatedat the level of mRNA expression in the inferior temporal cortex and inthe frontal cortex of the brains of patients with Alzheimer's disease(AD) relative to the brains of control individuals. It speculates thatCDK19 (CDC2L6) could be used as a drug target for the treatment of ADbut offers no evidence for that contention, aside from itsoverexpression in this disease.

US Patent Application Publication No. 20080033000 discloses a series ofstructurally related compounds, which inhibit the induction of all thetested genes by CDKI and also reverse CDKI-induced transcription. Thosemolecules showed little or no cytotoxicity in normal cells. As such,those molecules provided a promising starting point for developinguseful new compounds and methods for inhibiting the CDKI pathway.Greater potency of such molecules is still needed.

A compound inhibiting CDK8 and CDK19 preferentially to other CDKs hasbeen previously reported. This inhibitor is a steroidal alkaloidcortistatin A, isolated from the marine sponge Corticium simplex. Theonly biological properties reported for cortistatin A are its strong andhighly selective antiproliferative activity against human umbilical veinendothelial cells (HUVECs) and its ability to inhibit vascularendothelial growth factor (VEGF)-induced migration and tubular formationof HUVECs (Aoki et al., 2007). In particular, cortistatin A inhibitedthe proliferation of HUVECs with IC₅₀ of 1.8 nm, whereas itsantiproliferative activity against several other types of human cellswas 6-7 μM, or >3,000-fold higher than for HUVECs. Cee et al. (2009)tested cortistatin A at 10 mm for the ability to inhibit a panel of 402kinases (KinomeScan, Ambit Biosciences, San Diego, Calif.), and foundthat the strongest inhibition was observed for ROCK II (Percent ofControl (POC)=0), CDK19 (termed there CDK11) (POC=0.1), and CDK8(POC=0.95). The binding constants (Kd) were determined for CDK19 (Kd=10nm), CDK8 (Kd=17 nm), and ROCK I and II (Kd=250 nm and 220 nm,respectively). It has been unknown whether the selectiveantiproliferative effect of cortistatin A against HUVECs is due to theinhibition of CDK19, CDK8 or ROCK (Cee et al., 2009). Based on itsselective effect on endothelial cells, cortistatin A was proposed as anew type of anti-angiogenesis agent for the treatment of cancer (Aoki etal., 2007). However, the usefulness of anti-angiogenic agents forlong-term therapy of chronic diseases other than cancer is doubtful,since clinical experience with angiogenesis inhibitors such asbevacizumab (Avastin), has revealed severe side effects (Grothey andGalanis, 2009), such as gastrointestinal perforation, inhibition ofwound healing, and fatal pulmonary hemorrhage. Therefore, if anyinhibitors of CDK8 could be found that don't have the stronganti-proliferative effect on endothelial cells, characteristic forcortistatin A, such inhibitors could be used for many clinicalapplications where angiogenesis inhibitors are precluded by their sideeffects.

There is, therefore, a need for more potent compounds and methods forinhibiting the CDKI pathway which may have a variety of clinicalapplications in chemoprevention and therapy of different age-relateddiseases. There is also a need for more potent compounds and methods forinhibiting CDKI pathway-mediated paracrine support for cancerdevelopment by senescent fibroblasts and for inhibiting viralreplication. In addition there is a need for potent inhibitors of CDK8that do not have strong anti-proliferative effects on endothelial cells.

BRIEF SUMMARY OF THE INVENTION

The invention provides compounds, pharmaceutical formulations andmethods for treating degenerative diseases of the central nervous system(including Alzheimer's Disease and other dementias), cancer, viraldiseases, atherosclerosis, arthritis and chronic renal disease.

In a first aspect, the invention provides new compounds having enhancedpotency for inhibiting the induction of transcription by theCyclin-Dependent Kinase Inhibitor (CDKI) pathway. Initially, theinventors used a high throughput screening system, described in greaterdetail in application number PCT/US06/01046, to screen over 100,000drug-like small molecules from commercially available diversifiedcompound collections. Through this screening, the present inventorsidentified a set of active compounds. (See US Patent ApplicationPublication No. 20080033000.) These included a series of structurallyrelated compounds, which inhibit the induction of all the tested genesby CDKI and also reverse CDKI-induced transcription. Those moleculesshowed little or no cytotoxicity in normal cells. Those molecules didnot interfere with the cell cycle-inhibitory function of CDKIs and evenenhanced the induction of G1 cell cycle arrest by CDKI proteins. Suchcompounds also blocked the development of the senescent morphology infibroblasts arrested by DNA damage.

Based upon the above-described results, the present inventors have setout to develop new compounds that retain the benefits of thosepreviously identified compounds while providing even greater potency.

In a second aspect, the invention provides methods for enhancinginduction of G1 cell cycle arrest by CDKI proteins comprising contactinga cell with a compound that enhances the induction of G1 cell cyclearrest by CDKI proteins. In some preferred embodiments, the cellcycle-inhibitory activity of CDKI proteins is mediated by the inhibitionof CDK8. The enhancement of the induction of G1 cell cycle arrest byCDKI proteins can be used for the chemoprevention and treatment ofcancer and other diseases associated with abnormal cell proliferationand for increasing the ability of CDKI-inducing cancer therapeuticagents to arrest the growth of cancer cells. The method according to theinvention comprises contacting a cell with a small molecule compoundaccording to the invention.

In a third aspect the invention provides methods for inhibiting theproduction of tumor-promoting secreted factors by fibroblasts,comprising contacting the fibroblast with a compound according to theinvention.

In a fourth aspect, the compounds and methods according to the inventionare useful for treating a CDKI-mediated disease, including but notlimited to Alzheimer's disease, atherosclerosis, amyloidosis, arthritis,chronic renal disease, viral diseases and cancer. Thus, the inventionprovides a method for treating or therapeutically treating a mammalhaving a CDKI-mediated disease comprising administering to the mammal atherapeutically effective amount of a compound according to theinvention.

In a fifth aspect, the invention provides compounds that inhibit CDK8 toa greater extent than it inhibits certain other CDKs, and furtherinhibits CDK8 to a greater extent than it inhibits ROCK2.

In a sixth aspect, the invention provides methods for treating a mammalhaving a disease selected from degenerative diseases of the nervoussystem, including Alzheimer's Disease and other dementias, viraldiseases, atherosclerosis, arthritis and chronic renal disease, themethod comprising administering to the mammal a compound according tothe fifth aspect of the invention.

In a seventh aspect, the invention provides methods for treating amammal having a tumor that expresses β-catenin, the method comprisingadministering to the mammal a compound according to the fifth aspect ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures and IC₅₀ values for representative activecompounds according to the invention.

FIG. 2 shows the structures and IC₅₀ values for additionalrepresentative active compounds according to the invention.

FIG. 3 shows the effects of different doses of three active compounds onthe stimulation of the CMV promoter activity by p21, represented as GFPexpression from the CMV promoter in a reporter cell line carryingIPTG-inducible p21, normalized by cellular DNA content (a measure ofcell number) as measured by Hoechst 33342 staining, in the presence orin the absence of two concentrations of IPTG.

FIG. 4 shows results of an assay for paracrine antiapoptotic activity ofirradiated, senescent WI38 fibroblasts, as measured by the survival ofC8 cells in low-serum media, in which irradiated, senescent WI38fibroblasts were either untreated or treated with a compound accordingto the invention.

FIG. 5 shows inhibition of HIV-1 replication by a compound according tothe invention.

FIG. 6 shows structural formulae for compounds excluded from the claims.

FIG. 7 shows structural formulae for compounds 1, 20, 26, 43 and SNX-14.

FIG. 8 shows correlations of IC50 values for nine SNX2-class compounds,as determined by the inhibition of CMV-GFP induction by p21, with theeffect on the indicated kinases (% of control activity). The compoundswere tested at 10 μM concentrations. The IC50 values for two compoundsthat showed no activity at the highest tested concentration (40 μM) areplotted as 100 μM.

FIG. 9 shows results of shRNA analysis of the role of CDK8 and CDK19 inthe CKI pathway in HT1080 cells with IPTG-inducible p21 andp21-responsive CMV-GFP promoter.

-   -   A. QPCR measurements (in triplicate) of CDK8 and CDK19 mRNA        levels (arbitrary units) in cells that were either uninfected        (no shRNA) or infected with recombinant lentiviruses, carrying        either no insert (control) or shRNAs targeting CDK8 or CDK19,        and used for infection at the indicated dilutions of packaging        cell supernatant (1:8, 1:16 or 1:32).    -   B. FACS analysis of GFP expression in the same cells as in (A),        with or without 3-day treatment with 50 μM IPTG, expressed as        mean fluorescence intensity of the GFP-expressing live        (propidium iodide negative) cells.    -   C. The same analysis as in (B), with GFP fluorescence normalized        to cellular DNA amount (GFP/Hoechst) in microtiter wells assays        (similar to FIG. 3).    -   D. The same analysis as in (C), except that cells were untreated        or treated with 4 μM IPTG.

FIG. 10 shows effects of compound 1 on cell proliferation.

-   -   A. Effects of the indicated doses of compound 1 on the        proliferation of HUVECs; relative cell number measured by MTT        assay after 3 day exposure.    -   B. Effects of compound 1 on the proliferation of KB-3-1        carcinoma cells, measured as in (A).    -   C. Effects of compound 1 on proliferation of the indicated colon        carcinoma cell lines, measured by FACS quantitation of the        number of live (propidium iodide negative) cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the inhibition of the Cyclin-Dependent KinaseInhibitor (CDKI) pathway. More particularly, the invention relates tomethods for inhibiting the CDKI pathway for studies of and interventionin senescence-related diseases and cancer. The patents and publicationscited herein reflect the level of knowledge in this field and are herebyincorporated by reference in their entirety. Any conflict between theteachings of the cited references and this specification shall beresolved in favor of the latter.

The invention provides new compounds, pharmaceutical formulations andmethods for treating degenerative diseases of the central nervoussystem, including Alzheimer's Disease and other dementias, as well ascancer, and viral diseases.

The invention provides compounds and methods for inhibiting the CDKIpathway which may have a variety of clinical applications inchemoprevention and therapy of different age-related diseases. The CDKIpathway inhibitors methods according to the invention show little or nocytotoxicity in normal cells. These molecules do not interfere with thecell cycle-inhibitory function of CDKIs and even enhance the inductionof G1 cell cycle arrest by CDKI proteins. Compounds according to theinvention block the development of the senescent morphology infibroblasts arrested by DNA damage. They also inhibit the secretion ofanti-apoptotic factors by CDKI-arrested cells. In some instances, thecompounds of the invention are referred to herein as SNX2-classcompounds.

In a first aspect, the invention provides novel compounds that inhibitthe CDKI pathway. In certain embodiments, compounds according to theinvention are represented by the formula (I-A):

wherein R⁶ is selected from CN, F, Cl, Br, I, NO₂, CF₃, CHO, COR¹⁰,CO₂R¹⁰, CONR¹¹R¹², C(═S)NR¹¹R¹², C(═NR¹⁰)NR¹¹R¹², SOR¹⁰, SO₂R¹⁰ andSO₂NR¹¹R¹²,wherein R¹³ is 2-, 3-, or 4-H, F, Cl, Br, I, OH, OCH₃, CH₃, CF₃,NR¹¹R¹², CH₂R¹¹R¹², CO₂H and CONR¹¹R¹²;wherein R¹⁰ is H or C1-C6 alkyl, and each R¹¹ and R¹² is independently Hor C1-C6 alkyl, or R¹¹ and R¹² taken together form a ring;provided however, that when R⁶ is F then R¹³ is not 4-OH or 4-Cl, whenR⁶ is Cl then R¹³ is not 4-OH, 2-F, or 4-Cl, when R⁶ is Br then R¹³ isnot H, 4-OH, 4-SO₂NH₂, 4-OCH₃, 3-F, or 4-Cl, when R⁶ is I then R¹³ isnot 3-F or 2-Cl, and when R⁶ is NO₂ then R¹³ is not 3-F, 4-Cl, 4-Br,4-I, 2-OCH₃, 3-OCH₃, 4-OCH₃, 4-CONH₂, 4-CON(CH₃)₂, or 4-CO₂H.

In certain embodiments, compounds according to the invention arerepresented by the formula (I-B):

wherein R⁶ is selected from CN, F, Cl, Br, I, NO₂, CF₃, CHO, COR¹⁰,CO₂R¹⁰, CONR¹¹R¹², C(═S)NR¹¹R¹², C(═NR¹⁰)NR¹¹R¹², SOR¹⁰, SO₂R¹⁰ andSO₂NR¹¹R¹²,wherein R¹³ is 2-, 3-, or 4-H, F, Cl, Br, I, OH, OCH₃, CH₃, CF₃,NR¹¹R¹², CH₂R¹¹R¹², CO₂H and CONR¹¹R¹²;wherein R¹⁰ is H or C1-C6 alkyl, and each R¹¹ and R¹² is independently Hor C1-C6 alkyl, or R¹¹ and R¹² taken together form a ring.

In certain embodiments, compounds according to the invention arerepresented by the formula (I-C):

wherein R⁶ is selected from CN, F, Cl, Br, I, NO₂, CF₃, CHO, COR¹⁰,CO₂R¹⁰, CONR¹¹R¹², C(═S)NR¹¹R¹², C(═NR¹⁰)NR¹¹R¹², SOR¹⁰, SO₂R¹⁰ andSO₂NR¹¹R¹²,wherein R¹³ is 2-, 3-, or 4-H, F, Cl, Br, I, OH, OCH₃, CH₃, CF₃,NR¹¹R¹², CH₂R¹¹R¹², CO₂H and CONR¹¹R¹²;wherein R¹⁰ is H or C1-C6 alkyl, and each R¹¹ and R¹² is independently Hor C1-C6 alkyl, or R¹¹ and R¹² taken together form a ring.

In certain embodiments, compounds according to the invention arerepresented by the formula (II-A):

wherein R⁶ is selected from CN, F, Cl, Br, I, NO₂, CF₃, CHO, COR¹⁰,CO₂R¹⁰, CONR¹¹R¹², C(═S)NR¹¹R¹², C(═NR¹⁰)NR¹¹R¹², SOR¹⁰, SO₂R¹⁰ andSO₂NR¹¹R¹²,wherein R¹³ is 2-, 3-, or 4-H, F, Cl, Br, I, OH, OCH₃, CH₃, CF₃,NR¹¹R¹², CH₂R¹¹R¹², CO₂H and CONR¹¹R¹²;wherein R¹⁰ is H or C1-C6 alkyl, and each R¹¹ and R¹² is independently Hor C1-C6 alkyl, or R¹¹ and R¹² taken together form a ring;provided however, that when R⁶ is CHO then R¹³ is not H, when R⁶ is Clthen R¹³ is not 4-F or 4-OCH₃, when R⁶ is Br then R¹³ is not H, 2-Cl,4-Cl, 4-SO₂NH₂, 4-CO₂H, 4-OCH₃, or 4-CF₃, when R⁶ is I then R¹³ is notH, 2-CF₃ or 3-CF₃, and when R⁶ is NO₂ then R¹³ is not H or 4-Cl.

In certain embodiments, compounds according to the invention arerepresented by the formula (II-B):

wherein R⁶ is selected from CN, F, Cl, Br, I, NO₂, CF₃, CHO, COR¹⁰,CO₂R¹⁰, CONR¹¹R¹², C(═S)NR¹¹R¹², C(═NR¹⁰)NR¹¹R¹², SOR¹⁰, SO₂R¹⁰ andSO₂NR¹¹R¹²;wherein R¹⁰ is H or C1-C6 alkyl, and each R¹¹ and R¹² is independently Hor C1-C6 alkyl, or R¹¹ and R¹² taken together form a ring.

In certain embodiments, compounds according to the invention arerepresented by the formula (II-C):

wherein R⁶ is selected from F, Br, I, NO₂, CF₃, COR¹⁰, CO₂R¹⁰,CONR¹¹R¹², C(═S)NR¹¹R¹², C(═NR¹⁰)NR¹¹R¹², SOR¹⁰, SO₂R¹⁰ and SO₂NR¹¹R¹²;wherein R¹⁰ is H or C1-C6 alkyl, and each R¹¹ and R¹² is independently Hor C1-C6 alkyl, or R¹¹ and R¹² taken together form a ring.

In certain embodiments, compounds according to the invention arerepresented by the formula (III):

wherein R⁶ is selected from CN, F, Cl, NO₂, CF₃, CHO, COR¹⁰, CO₂R¹⁰,CONR¹¹R¹², C(═S)NR¹¹R¹², C(═NR¹⁰)NR¹¹R¹², SOR¹⁰, SO₂R¹⁰ and SO₂NR¹¹R¹²;wherein R¹⁰ is H or C1-C6 alkyl, and each R¹¹ and R¹² is independently Hor C1-C6 alkyl, or R¹¹ and R¹² taken together form a ring.

In certain embodiments, compounds according to the invention arerepresented by the formula (IV-A):

wherein R⁶ is selected from CN, F, Cl, Br, I, NO₂, CF₃, CHO, COR¹⁰,CO₂R¹⁰, CONR¹¹R¹², C(═S)NR¹¹R¹², C(═NR¹⁰)NR¹¹R¹², SOR¹⁰, SO₂R¹⁰ andSO₂NR¹¹R¹²;wherein R¹³ is 2-, 3-, or 4-H, F, Cl, Br, I, OH, OCH₃, CH₃, CF₃,NR¹¹R¹², CH₂R¹¹R¹², CO₂H and CONR¹¹R¹²;wherein R¹⁰ is H or C1-C6 alkyl, and each R¹¹ and R¹² is independently Hor C1-C6 alkyl, or R¹¹ and R¹² taken together form a ring; andwherein n is from 1-3;provided however that when R⁶ is Br, I or NO₂ then R¹³ is not H.

In certain embodiments, compounds according to the invention arerepresented by the formula (IV-B):

wherein R⁶ is selected from CN, F, Cl, Br, I, NO₂, CF₃, CHO, COR¹⁰,CO₂R¹⁰, CONR¹¹R¹², C(═S)NR¹¹R¹², C(═NR¹⁰)NR¹¹R¹², SOR¹⁰, SO₂R¹⁰ andSO₂NR¹¹R¹²;wherein R¹³ is 2-, 3-, or 4-H, F, Cl, Br, I, OH, OCH₃, CH₃, CF₃,NR¹¹R¹², CH₂R¹¹R¹², CO₂H and CONR¹¹R¹²;wherein R¹⁰ is H or C1-C6 alkyl, and each R¹¹ and R¹² is independently Hor C1-C6 alkyl, or R¹¹ and R¹² taken together form a ring; andwherein n is from 1-3.

In certain embodiments, compounds according to the invention arerepresented by the formula (V):

wherein each of X¹, X² and X³ is independently N or CH, and one, butonly one, of X¹, X² and X³ is N;wherein R⁶ is selected from CN, F, Cl, Br, I, NO₂, CF₃, CHO, COR¹⁰,CO₂R¹⁰, CONR¹¹R¹², C(═S)NR¹¹R¹², C(═NR¹⁰)NR¹¹R¹², SOR¹⁰, SO₂R¹⁰ andSO₂NR¹¹R¹²;wherein R¹³ is 2-, 3-, or 4-H, F, Cl, Br, I, OH, OCH₃, CH₃, CF₃,NR¹¹R¹², CH₂R¹¹R¹², CO₂H and CONR¹¹R¹²;wherein R¹⁰ is H or C1-C6 alkyl, and each R¹¹ and R¹² is independently Hor C1-C6 alkyl, or R¹¹ and R¹² taken together form a ring; andwherein n is from 1-3;provided however, that when X¹ is N and n=1, then R⁶ is not Br, when X²is N and n=1 then R⁶ is not Br, I or CHO and when X³ is N and n=1 thenR⁶ is not Br or CHO.

In certain preferred embodiments the compounds according to theinvention have the structures shown in FIG. 1 or FIG. 2.

In a second aspect, the invention provides methods for enhancinginduction of G1 cell cycle arrest by CDKI proteins comprising contactinga cell with a compound having the structure (I-A), (I-B), (I-C), (II-A),(II-B), (II-C), (III), (IV-A), (IV-B), (V) or (VI):

wherein R⁴ is selected from C1-C6 alkyl and C₁₋₃—R¹⁴, wherein R¹⁴ is a 6membered aryl or heteroaryl group or a fused bicyclic aryl or heteroarylgroup, either of which may be optionally substituted with one or moresubstituent selected from 2-, 3-, or 4-H, F, Cl, Br, I, OH, OCH₃, CH₃,CF₃, NR¹¹R¹², CH₂R¹¹R¹², CO₂H and CONR¹¹R¹², and wherein R⁶ is anelectron withdrawing group. In certain embodiments, the electronwithdrawing group is selected from CN, F, Cl, Br, I, NO₂, CF₃, CHO,COR¹⁰, CO₂R¹⁰, CONR¹¹R¹², C(═S)NR¹¹R¹², C(═NR¹⁰)NR¹¹R¹², SOR¹⁰, SO₂R¹⁰and SO₂NR¹¹R¹², wherein R¹⁰ is H or C1-C6 alkyl, and each R¹¹ and R¹² isindependently H or C1-C6 alkyl, or R¹¹ and R¹² taken together form aring. In certain preferred embodiments, the small molecule has astructure selected from the group of structures shown in FIG. 1 or 2.

In a third aspect the invention provides methods for inhibiting theproduction of tumor-promoting secreted factors by fibroblasts,comprising contacting the fibroblast with a compound having thestructure (I-A), (I-B), (I-C), (II-A), (II-B), (II-C), (III), (IV-A),(IV-B), (V) or (VI), including without limitation the compounds shown inFIG. 1 or 2. In certain embodiments, the fibroblast is in a mammal,including a human.

In a fourth aspect of the invention, the invention provides a method fortreating or therapeutically treating a mammal having a CDKI-mediateddisease comprising administering to the mammal an effective ortherapeutically effective amount of a compound having the structure(I-A), (I-B), (I-C), (II-A), (II-B), (II-C), (III), (IV-A), (IV-B), (V)or (VI), including without limitation the compounds shown in FIG. 1 or2. Preferred CDKI-mediated diseases include, without limitation,Alzheimer's disease, other dementias, amyloidosis, atherosclerosis,renal disease, viral diseases, and cancer. Preferred mammals include ahuman.

In certain embodiments the viral disease is human immunodeficiency virus(HIV) infection.

In a fifth aspect, the invention provides compounds that inhibit CDK8 toa greater extent than they inhibit certain other CDKs, and furtherinhibit CDK8 to a greater extent than they inhibit ROCK2. Such moleculesare small molecules and do not include large molecules such as antisenseoligonucleotides, siRNA, ribozymes, or proteins. In some embodiments,such compounds further inhibit CDK8 to a greater extent than ROCK1. Incertain embodiments, the compound inhibits CDK8 to a greater extent thanit inhibits CDK1. In certain embodiments, the compound inhibits CDK8 toa greater extent than it inhibits CDK1, CDK2, CDK4 and CDK6. In certainembodiments, the compound further inhibits CDK8 to a greater extent thanCDK5, CDK7 and CDK9. In preferred embodiments, such greater extent is atleast 2-fold, both for either set of CDKs and for ROCK2. In someembodiments, the compound further inhibits CDK8 to a greater extent thanROCK1, preferably by at least 2-fold. In some embodiments the comparisonof inhibition of CDK8 with inhibition of ROCK2 or ROCK1 is carried outat a concentration of 10 μM compound. Extent of inhibition is measuredby the assays taught in the Examples in this specification, includingthe assay conditions employed by the service providers utilized herein.Results of these assays are commonly expressed herein as percent ofcontrol (POC), with the control being no compound being present.

In a sixth aspect, the invention provides methods for treating ortherapeutically treating a mammal having a disease selected fromdegenerative diseases of the nervous system, including Alzheimer'sDisease and other dementias, viral diseases, atherosclerosis, arthritisand chronic renal disease, the method comprising administering to themammal an effective amount or a therapeutically effective amount of acompound according to the fifth aspect of the invention.

In a seventh aspect, the invention provides methods for treating ortherapeutically treating a mammal having a tumor that expressesβ-catenin, the method comprising administering to the mammal aneffective amount or a therapeutically effective amount of compoundaccording to the fifth aspect of the invention.

Pharmaceutical Formulations and Administration

In the methods according to the invention, the compounds described abovemay be incorporated into a pharmaceutical formulation. Such formulationscomprise the compound, which may be in the form of a free acid, salt orprodrug, in a pharmaceutically acceptable diluent, carrier, orexcipient. Such formulations are well known in the art and aredescribed, e.g., in Remington's Pharmaceutical Sciences, 18th Edition,ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990.

The characteristics of the carrier will depend on the route ofadministration. As used herein, the term “pharmaceutically acceptable”means a non-toxic material that is compatible with a biological systemsuch as a cell, cell culture, tissue, or organism, and that does notinterfere with the effectiveness of the biological activity of theactive ingredient(s). Thus, compositions according to the invention maycontain, in addition to the inhibitor, diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials well known inthe art.

As used herein, the term pharmaceutically acceptable salts refers tosalts that retain the desired biological activity of theabove-identified compounds and exhibit minimal or no undesiredtoxicological effects. Examples of such salts include, but are notlimited to, salts formed with inorganic acids (for example, hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, andthe like), and salts formed with organic acids such as acetic acid,oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid,benzoic acid, tannic acid, palmoic acid, alginic acid, polyglutamicacid, naphthalenesulfonic acid, naphthalenedisulfonic acid,methanesulfonic acid, p-toluenesulfonic acid and polygalacturonic acid.The compounds can also be administered as pharmaceutically acceptablequaternary salts known by those skilled in the art, which specificallyinclude the quaternary ammonium salt of the formula —NR+Z—, wherein R ishydrogen, alkyl, or benzyl, and Z is a counterion, including chloride,bromide, iodide, —O-alkyl, toluenesulfonate, methylsulfonate, sulfonate,phosphate, or carboxylate (such as benzoate, succinate, acetate,glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate,cinnamoate, mandeloate, benzyloate, and diphenylacetate).

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount without causing serious toxic effectsin the patient treated. A “therapeutically effective amount” is anamount sufficient to alleviate or eliminate signs or symptoms of thedisease. The effective dosage range of the pharmaceutically acceptablederivatives can be calculated based on the weight of the parent compoundto be delivered. If the derivative exhibits activity in itself, theeffective dosage can be estimated as above using the weight of thederivative, or by other means known to those skilled in the art. Incertain applications, including without limitation, senile dementiassuch as Alzheimer's, an effective dose range for a 70 kg patient is fromabout 50 mg per patient per day up to about 10 grams per patient perday, or the maximum tolerated dose. In certain preferred embodiments thedose range is from about 200 mg per patient per day to about 10 g perpatient per day. In certain preferred embodiments the dose range is fromabout 200 mg per patient per day to about 5 g per patient per day. Thedose in each patient may be adjusted depending on the clinical responseto the administration of a particular drug.

Administration of the pharmaceutical formulations in the methodsaccording to the invention may be by any medically accepted route,including, without limitation, parenteral, oral, sublingual,transdermal, topical, intranasal, intratracheal, or intrarectal. Incertain preferred embodiments, compositions of the invention areadministered parenterally, e.g., intravenously in a hospital setting. Incertain other preferred embodiments, administration may preferably be bythe oral route.

The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not intended to limit thescope of the invention.

Example 1 Determination of IC₅₀ Values for CDKI-Pathway InhibitorCompounds

The lead optimization series compounds have been tested for the abilityto prevent the induction of transcription by p21 in HT1080 fibrosarcomacells, as described in US20080033000. Briefly, the reporter cell linedescribed in the above-referenced application, which carries IPTG(isopropyl-β-thio-galactoside)-inducible p21 gene together with ap21-inducible promoter driving the expression of Green FluorescentProtein (GFP) was plated in 96-well plates, at 2000 cells per well forthe no-IPTG arm of the assay and at 5000 cells per well for the IPTG armof the assay. To induce the maximal levels of p21, IPTG was added to 50μM concentration. Serial 2-fold dilutions of the tested compounds wereadded in quadruplicates to wells with and without IPTG, ˜10 min afterIPTG addition. After culturing under standard cell culture conditionsfor 72 hours, GFP fluorescence and Hoechst 33342 staining of cellularDNA (a measure of relative cell number) were measured as described inthe previous application US20080033000, and IC50 values were calculatedfor each compound. The results of this analysis are presented in FIGS. 1and 2.

To determine IC₅₀ values of the compounds at lower, more physiologicallevels of p21 induction, assays with selected compounds were repeated asdescribed above, except that p21 was induced by a lower, 8 μMconcentration of IPTG. The results of these assays for three activecompounds of SNX2 family are shown in FIG. 3. The IC₅₀ values calculatedin this assay are 570 nM for compound 1,600 nM for compound 14 and 1.85μM for compound 3. These values are ˜2-fold lower than the correspondingvalues determined for the same compounds in assays utilizing 50 μM IPTG,consistent with the different levels of p21 induced in two types ofassays. The activities of compounds 1 and 14 exceed those of the mostactive of the previously disclosed compounds of US Patent ApplicationPublication No. 20080033000 by an order of magnitude.

Example 2 Inhibition of Tumor-Promoting Activity of Irradiated,Senescent Fibroblasts

As discussed by Roninson (Cancer Lett. 179, 1-14, 2002), all thetreatments known to induce the production of tumor-promoting secretedfactors by cancer-associated stromal fibroblasts activate the CDKIpathway, including exposure to ionizing radiation and the induction ofsenescence. Since this pathway is the target of compounds according tothe invention, such compounds may be the first agents to targettumor-supporting activities of cancer-associated fibroblasts. Some ofthe fibroblast-activating treatments include exposure to ionizingradiation and the induction of senescence through various means(Krtolica et al., Proc. Natl. Acad. Sci. USA 98, 12072, 2001). We haveadopted a 96-well assay for anti-apoptotic activity that we havepreviously used with p21-expressing fibrosarcoma cells (Chang et al.,Proc. Natl. Acad. Sci. USA 97, 4291-4296, 2000), to measure atumor-promoting activity of normal proliferating fibroblasts andfibroblasts exposed to ionizing radiation and consequently developingthe senescent phenotype. In this assay, WI38 fibroblasts are irradiatedwith 10 Gy using an MDS Nordion Gammacell 40 irradiator, or leftunirradiated. The irradiated WI38 cells in suspension are added intriplicate to a 6-well tissue culture plate, at 250,000 cells per well,with and without the test compounds (used either at a single dose or ata series of concentrations from 0.3-10 μM). Unirradiated WI38 cells areplated at 125,000 cells per well, allowing them to reach the samedensity after growth over the time of the assay. On the third day, anapoptosis-sensitive murine transformed cell line C8, expressing fireflyluciferase, is added to each well at 55,000 cells/well, and the cellmixtures are cultured overnight under standard conditions. On the nextday (day 4), the media are replaced with media containing low (0.5%) FC2serum and cultured for 3 days. On day 7, cells are lysed and the amountof luciferase activity associated with C8 cells is determined.

The results of such an assay carried out with compound 1 are shown inFIG. 4. In this experiment, co-culture with irradiated/senescent WI38fibroblasts protects apoptosis-prone C8 cells from death under low serumculture conditions; the effect of irradiated WI38 fibroblasts is 6-foldhigher than the corresponding effect of non-senescent WI38 cells.However, the addition of compound 1 greatly diminished this paracrinetumor-promoting effect of the irradiated/senescent fibroblasts (up toalmost 3-fold). Hence, compounds according to the invention can inhibitproduction of tumor-supporting secreted factors by fibroblasts,indicating their utility in the treatment of different types of solidtumors.

Example 3 Inhibition of HIV Replication

The following assay was carried out by James McSharry at Ordway ResearchInstitute at the request of Igor Roninson. HIV replication was assayedin a cell-to-cell model of HIV-1 transmission using chronically infectedH9_(IIIB) as donor cells cocultivated with uninfected CEM-ss leukemiacell line as recipient cells. 1 ml of RPMI1640 medium (+10% Fetal BovineSerum+Penicillin/Streptomycin+glutamine) containing 10⁴ H9_(IIIB) cellswas mixed with 1 ml of the same medium containing 10⁶ CEM-ss cells. Themixture was spun at 1500 rpm for 5 min, the supernatant was aspirated,and cell pellets were suspended in 10 ml of the same media containingthe following final concentrations of compound 1: 5, 2.5, 1.25, 0.625,0.3125 and 0 μM (diluted from a stock solution of 500 μM in 25% DMSO).The suspended cells were placed into a 25 cm² flask and incubated at 37°C., 5% CO₂. On day 3, the cells were counted with a light microscope andan hemocytometer after trypan blue staining, which was used to determinethe fraction of viable (trypan blue negative) cells. On day 5, thefraction of HIV-infected cells was determined using the K57-FITCconjugated monoclonal antibody to the HIV p24 antigen. For thismeasurement, the cells in each flask were mixed, 1 ml of cell culturewas removed from each flask and placed into round bottom centrifugetubes. The cells were separated from the medium by centrifugation at1500 RPM for 5 minutes, the supernatants were aspirated and discardedand the cell pellets suspended in a fixative. After 15 min at roomtemperature, the cell pellets were washed with 3 ml of PBS, the cellswere pelleted at 1500 RPM for 5 min, the supernatants discarded and thepellets permeabilized and then treated with the anti-p24 antibody. Aftera 15 minute incubation at room temperature in the dark, the monoclonalantibody was washed off with 3 ml of PBS and the cells collected bycentrifugation at 1500 RPM for 5 min. The final pellets were suspendedin 0.5 ml of PBS and the percentage of HIV p24-positive cells wasdetermined by FACS analysis.

The results of these assays are shown in FIG. 5. The addition ofcompound 1 produced a strong dose-dependent decrease in the fraction ofHIV-infected (p24-positive) cells, with the calculated EC50 of 0.58 μM.Thus, the CKI pathway inhibitor is capable of inhibiting viral (HIV)replication.

Example 4 Selective Inhibition of CDK8 and CDK19 by SNX2-Class Compounds

FIG. 7 shows the structures of six specific SNX2-class compounds,including SNX14, the most potent pre-existing structure of SNX2 classdisclosed in US Patent Application 2009/0281129, as well as compounds 1,14, 20, 26, and 43 created by the instant inventors and disclosed inU.S. Patent Application 61/264,991. Table 1 shows the IC₅₀ values forthe activities of these compounds in a cellular assay for CDKI pathwayinhibition, which is described in our previous patent application. Thisassay is based on the induction of green fluorescent protein (GFP)expression from the cytomegalovirus (CMV) promoter in human HT1080fibrosarcoma cells that express CDKI p21 from anisopropyl-(3-thio-galactoside (IPTG)-inducible promoter. The compoundactivity as a CDKI pathway inhibitor is measured by its ability toprevent the stimulation of the CMV promoter upon the addition ofp21-inducing IPTG. CMV promoter activity is measured by the ratio of GFPfluorescence to the relative cell number, as determined by stainingcellular DNA with Hoechst 33342. This assay was conducted in two modes.In the first mode p21 is induced to the maximal level, using 50 μM IPTG.In the second mode, lower, more physiological levels of p21, are inducedby a lower IPTG concentration (2 μM). The IC₅₀ values are presented inTable 1, where the tested compounds show 2-3 times higher potency (lowerIC₅₀) in the 2-μM IPTG than in the 50-μM IPTG assay.

TABLE 1 Activities of some SNX2-class compounds. IC₅₀ or Kd (nM) Com-Com- Com- Com- Com- Assay pound pound pound pound pound Compound SNX14 120 26 43 14 Cell-based 6430 641 199 769 220 2540 assay (IC₅₀) CDK8 10000830 240 570 240 2500 inhibition (Kd) CDK19 3800 310 99 160 190 1100inhibition (Kd)

SNX2-class compounds are 4-aminoquinazolines, and such structures are apart of the pharmacophore for several protein kinase inhibitors, such asEGF receptor inhibitor gefitinib (Notably, we have tested gefitinib inour assays and found it inactive for CDKI pathway inhibition). Thissimilarity led us to test SNX14 for the ability to inhibit 285 kinases,using the services of KinaseProfiler of Millipore Corporation (285kinases). Subsequently, compound 1 was tested for the inhibition of alarger panel of 442 kinases, using the services of KinomeScan, adivision of Ambit Biosciences, San Diego, Calif. The KINOMEscan assayhas been described in detail in Fabian et al., Nat. Biotechnol. 23: 329(2005) and Karaman et al., Nat. Biotechnol. 26: 127 (2008).

The KinomeScan assay is based on a competition binding assay thatquantitatively measures the ability of a compound to compete with animmobilized, ATP site directed ligand. The assay is performed bycombining three components: DNA-tagged kinase; immobilized ligand; and atest compound. The ability of the test compound to compete with theimmobilized ligand is measured via quantitative PCR of the DNA tag. Thecompounds (tested at 10 μM) showed significant inhibition of a number ofkinases, with the more potent compound 1 showing the strongestinhibition (>99%) for CDK19 (0.05 percent of control (POC) binding) andCDK8 (0.25 POC) in the KinomeScan assay. No other members of the CDKfamily were inhibited to a comparable degree. The CDKs tested byMillipore for inhibition with SNX14 included CDK1, CDK2, CDK3, CDK5,CDK7 and CDK9; none of these were inhibited to less than 49 percent ofcontrol (POC) activity. The CDKs tested by KinomeScan for the effect ofCompound 1 included CDK2, CDK3, CDK4, CDK5, CDK7, CDK8, CDK9 and CDK19;aside from CDK8 and CDK19, the strongest inhibited CDK was CDK7 (33% POCbinding). To determine the inhibition of which kinases correlates withthe biological activity of SNX2-class compounds, we tested eightadditional compounds of the SNX2 family, which gave different IC₅₀values in the cellular assays, for the ability to affect 24 kinases thatwere most susceptible to compound 1, through the services of KinomeScan.Table 2 shows the results of this analysis, where the effect on thecorresponding kinases is expressed as POC for the ligand binding by thecorresponding enzymes in the presence of 10 μM of each of the testedcompounds, together with IC₅₀ values for the corresponding compounds asdetermined in the cellular assay. (The IC₅₀ values shown for SNX14 andcompounds 1 and 20 in Table 2 differ from the corresponding values inTable 1 not only because they were determined in separate assays, butalso because the IC₅₀ values in Table 2 were calculated on theassumption that the maximal effect of a compound corresponds to zeroGFP/Hoechst value, whereas the IC₅₀ values in Table 1 are calculated onthe basis of the maximal inhibition produced by the highest doses of thetested compound.) FIG. 4 plots IC₅₀ values versus POC for seven of themost sensitive kinases in Table 2. CDK8 and CDK19, but not the othertested kinases, showed excellent correlation between IC₅₀ in thecellular assay and POC in the kinase assay (R²=0.96 and 0.99,respectively). Both CDK8 and CDK19 were inhibited >98% by five of themost potent compounds tested in this assay, except for compound 26 (FIG.2), which affected CDK8 by almost 100% but CDK19 by 94.6%; all the othercompounds affected CDK19 more than CDK8. Compound 26 also shows higherselectivity than the other tested compounds for CDK8 and CDK19 relativeto the other tested kinases. We have also determined the bindingconstant (Kd) for SNX14, compound 1, compound 20, compound 26, compound43 and compound 14 in kinase assays for CDK8 and CDK19 (Table 1).

TABLE 2 Inhibition of different kinases by SNX2-class compounds.Compound 1 20 26 43 14 44 45 46 47 IC50 1.3 0.6 1.15 0.95 1.2 1112.8 >100 >100 Kinase CDK19 0.05 0 5.6 0.25 0.1 9.2 21 100 100 CDK8 0.750.15 0 1.6 0.4 24 37 100 100 PIP5K2C 1.2 2.4 39 4 16 59 60 61 65 CLK23.8 16 58 9.6 24 34 8.6 66 58 CLK1 4.2 12 32 9 18 17 8.4 48 84 STK16 4.225 16 30 2.4 70 17 80 100 YSK4 5.4 31 44 29 21 39 41 91 79 CLK4 8 11 269.6 16 17 7.8 24 100 PIK3CG 8 40 58 35 35 88 82 94 98 IRAK1 8.2 26 897.6 35 64 25 81 100 SLK 11 61 73 92 90 43 59 88 100 MAP4K2 12 21 28 2816 35 12 82 66 EGFR (G719C) 13 34 68 53 92 0.85 59 61 55 BTK 15 100 100100 100 100 100 81 100 CIT 15 7.9 40 14 9.8 18 50 86 90 MAPKAPK5 15 92100 85 100 94 100 91 95 CSNK1G2 16 62 57 53 43 80 53 55 72 CSNK1G3 16 8250 54 52 84 50 100 56 PIK3CA (I800L) 17 73 81 87 62 86 83 83 58 HIPK2 1828 73 30 34 62 20 30 78 HIPK3 18 28 66 42 35 53 28 34 75 MEK3 18 76 7864 29 72 71 100 100 MEK5 18 62 61 41 13 59 46 17 36 FLT3 (D835Y) 19 6955 76 32 55 4.7 19 93

Example 5 Mediation of the CDKI Pathway by CDK8, but not by CDK19

To test the role of CDK19 and CDK8 in the CDKI pathway, we havedecreased the expression of these genes through RNA interference (RNAi)using lentiviral vectors expressing short hairpin RNA (shRNA) targetingthe corresponding genes and cloned in the pLKO.1 vector backbone (OpenBiosystems, Huntsville, Ala.). The CDK8 mRNA sequence targeted by thecorresponding shRNA is GCCCUUAUCAAGUAUAUAUGGAAA, [SEQ ID NO.: 1] and theshRNA target sequence in CDK19 mRNA is AGGACUGAUAGCUCUUCUUUA [SEQ IDNO.: 2]. Control LKO.1 lentivirus (carrying the puromycin resistancemarker) and lentiviruses expressing CDK8 and CDK19 shRNAs, alone or incombination, were generated by transfection with ViraPower lentiviralpackaging mix into 293FT cells (Invitrogen), and packaged virus wastransduced into the same HT1080-based reporter cell line that was usedin the assays shown in FIG. 3. Cell populations, transduced with threedifferent dilutions of the lentivirus-containing supernatant from thetransfected packaging cells (1:8, 1:16 and 1:32), were selected with 2m/ml puromycin and tested for the knockdown of CDK8 and CDK19 mRNA byquantitative reverse transcription-PCR, using the following PCR primers:for CDK8, TGGGATTTCCTGCAGATAAAGATTGGG [SEQ ID NO.: 3] andAGGGGTCCTGCATAGCCTGTT [SEQ ID NO.: 4]; for CDK 19,ACACAAGGTCAAGCCTGACAGCA [SEQ ID NO.: 5] and TGGAATCTGGCAGCCGGCAA [SEQ IDNO.: 6]. As shown in FIG. 9A, CDK8 targeting shRNA decreased CDK8 mRNAlevel by 65-80% and had no effect on CDK19 mRNA levels, and CDK19targeting shRNA decreased CDK19 mRNA level by ˜75% and had no effect onCDK8 mRNA levels. Co-transduction with CDK8 and CDK19-targeting shRNAhad a moderate effect on both mRNAs (35-65% knockdown) (FIG. 9A). Wethen assayed the untransduced and lentivirus-transduced cell populationsfor CMV-GFP expression, in the presence and in the absence of IPTG(which induces p21 expression). Two types of assays were used for thisanalysis. The first assay was the same 96-well plate assay, whichmeasures GFP fluorescence normalized by DNA content; the assays wereconducted conducted using either 501.1M IPTG (FIG. 9B) or 41.1M IPTG(FIG. 9C), providing for different levels of p21 induction. The secondassay was a flow cytometric assay, where GFP expression was measured byfluorescence using FACS, and dead cells (as defined by propidium iodideuptake) or cells that lost GFP expression were excluded from theanalysis. The mean fluorescence intensity of live GFP-positive cells ineach population is plotted in FIG. 9D. Both assays produced the sameresults: the knockdown of CDK8 alone or of both CDK8 and CDK19 decreasedIPTG-induced CMV-GFP expression, whereas the knockdown of CDK19 alonenot only failed to decrease such expression but in fact moderatelyincreased it in the absence of IPTG. The opposite effects of CDK8 andCDK19 knockdown in our system parallel the findings of Tsutsui et al.(2008) who found through siRNA knockdown assays that CDK8 is a positiveregulator but CDK19 is a negative regulator of viral activatorVP16-dependent transcription. Hence, CDK8 but not CDK19 is the target ofSNX2-class compounds responsible for their activity as CDKI pathwayinhibitors.

Example 6 Comparison of Kinase Inhibition and Cell Line InhibitionProfiles of Cortistatin A and SNX2-Class Compounds

Table 3 shows the data on the effects of cortistatin A (the onlypublished compound that inhibits CDK8 and CDK19 preferentially to otherCDKs) on the activity of 15 kinases that were the most sensitive to thiscompound. The data from Cee et al. (2009), expressed as POC in thepresence of 10 μM cortistatin A, are shown next to our data on theeffects of 10 μM compound 1 on the same kinases; both Cee et al. (2009)and we used the same kinome profiling service (KinomeScan). It isapparent from Table 3 that, aside from the inhibition of CDK19 and CDK8,the kinase sensitivity profiles of the two compounds are very different.In particular, cortistatin A decreased ROCK2 binding to 0 POC % andROCK1 binding to 21 POC, whereas the corresponding values for compound 1were 89 POC and 79 POC, respectively.

TABLE 3 Kinase inhibition by cortistatin A and SNX2-class compound. POCKinase Cortistatin A Compound 1 ROCK2 0 89 CDK19 0.1 0.05 CDK8 0.95 0.75LTK 2.9 100 ALK 4.4 78 PIM2 4.4 52 PKACa 8.7 82 PKACb 13 89 MET 18 86PRKG2 21 85 RIOK2 21 23 ROCK1 21 79 CLK4 26 8 ROS1 26 63 CIT 28 15 JNK129 45

If the biological activity of cortistatin A as a highly selectiveinhibitor of HUVEC proliferation is due to the inhibition of CDK8 and/orCDK19, then a SNX2-class compound would be expected to show the sameselective inhibition of HUVECs. If, on the other hand, HUVEC inhibitionis mediated by another activity of cortistatin A, such as ROCKinhibition, a SNX2-class compound should not display a similarselectivity for HUVECs. To test the growth inhibitory effects ofcompound 1 on HUVECs, we have obtained HUVECs from Lifeline CellTechnology (Oceanside, Calif.) and analyzed the effects of differentdoses of compound 1 on HUVEC proliferation, in a 3-day MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay(FIG. 10A). Compound 1 exhibited a moderate growth-inhibitory effect onHUVEC proliferation, with IC50 of 10.4 μM. Notably, compound 1concentrations that largely inhibited the CKI pathway, such as 0.625 or1.25 μM, had little or no effect on HUVEC cell number relative tountreated control. Hence, SNX2-class compounds can inhibit the CKIpathway at the concentrations where they do not affect endothelial cellproliferation. We have also determined the effect of compound 1 on theproliferation of KB-3-1 carcinoma cells, which is one of the cell linestested by Aoki et al. (2007) for growth inhibition by cortistatin A(FIG. 10B). This analysis yielded IC50 of 44.2 μM, which is only4.3-fold higher than the IC50 for HUVEC cells, as opposed to the3,900-fold differential for cortistatin A (Aoki et al., 2007). Hence,SNX2-class compounds do not have the selective endothelial cellinhibitory activity of cortistatin A. It seems most likely thatinhibition of endothelial cell proliferation by cortistatin A ismediated by the inhibition of ROCK (which is not inhibited by SNX2-classcompounds), since ROCK inhibitors fasudil and Y-27632 were shown toinhibit VEGF-stimulated endothelial cell proliferation, migration, andtube formation (references provided in Cee et al., 2009).

Example 7 Inhibition of Colon Carcinoma Cell Growth by SNX2-ClassCompounds

Inhibition of β-catenin, which is positively regulated by CDK8, wasreported to inhibit proliferation of some colon carcinoma cell lines(Firestein et al., 2008). We have therefore determined the ability ofcompound 1 to inhibit the proliferation of several colon carcinoma celllines, including human DLD1, HCT116, SW480 and murine C26. This analysiswaqs carried out by incubating cells for 72 hrs in the presence of 0,2.5, 5 or 10 μM compound 1, followed by FACS measurement of the numberof live cells, as defined by the lack of uptake of membrane-impermeabledye propidium iodide. The results shown in FIG. 10C demonstrate thatcompound 1 inhibited the growth of all four colon carcinoma cell lines,with the strongest growth-inhibitory effect (IC₅₀ ˜5 μM) found for DLD1cells, proliferation of which is notably sensitive to β-catenininhibition (Firestein et al., 2008). This result indicates the utilityof CDK8-inhibiting SNX2-class compounds as inhibitors of the growth oftumors expressing β-catenin.

REFERENCES Reference List

-   Alarcon, C., Zaromytidou, A. I., Xi, Q., Gao, S., Yu, J., Fujisawa,    S., Barlas, A., Miller, A. N., Manova-Todorova, K., Macias, M. J.,    Sapkota, G., Pan, D., and Massague, J. (2009). Nuclear CDKs drive    Smad transcriptional activation and turnover in BMP and TGF-beta    pathways. Cell 139, 757-769.-   Aoki, S., Watanabe, Y., Tanabe, D., Arai, M., Suna, H., Miyamoto,    K., Tsujibo, H., Tsujikawa, K., Yamamoto, H., and Kobayashi, M.    (2007). Structure-activity relationship and biological property of    cortistatins, anti-angiogenic spongean steroidal alkaloids. Bioorg.    Med. Chem. 15, 6758-6762.-   Cee, V. J., Chen, D. Y., Lee, M. R., and Nicolaou, K. C. (2009).    Cortistatin A is a high-affinity ligand of protein kinases ROCK,    CDK8, and CDK11. Angew. Chem. Int. Ed Engl. 48, 8952-8957.-   Donner, A. J., Szostek, S., Hoover, J. M., and Espinosa, J. M.    (2007). CDK8 is a stimulus-specific positive coregulator of p53    target genes. Mol. Cell. 27, 121-133.-   Firestein, R., Bass, A. J., Kim, S. Y., Dunn, I. F., Silver, S. J.,    Guney, I., Freed, E., Ligon, A. H., Vena, N., Ogino, S., Chheda, M.    G., Tamayo, P., Finn, S., Shrestha, Y., Boehm, J. S., Jain, S.,    Bojarski, E., Mermel, C., Barretina, J., Chan, J. A., Baselga, J.,    Tabernero, J., Root, D. E., Fuchs, C. S., Loda, M., Shivdasani, R.    A., Meyerson, M., and Hahn, W. C. (2008). CDK8 is a colorectal    cancer oncogene that regulates beta-catenin activity. Nature 455,    547-551.-   Firestein, R. and Hahn, W. C. (2009). Revving the Throttle on an    oncogene: CDK8 takes the driver seat. Cancer Res 69, 7899-7901.-   Grothey, A. and Galanis, E. (2009). Targeting angiogenesis: progress    with anti-VEGF treatment with large molecules. Nat. Rev. Clin.    Oncol. 6, 507-518.-   Malumbres, M., Harlow, E., Hunt, T., Hunter, T., Lahti, J. M.,    Manning, G., Morgan, D. O., Tsai, L. H., and Wolgemuth, D. J.    (2009). Cyclin-dependent kinases: a family portrait. Nat. Cell Biol.    11, 1275-1276.-   Morris, E. J., Ji, J. Y., Yang, F., Di Stefano, L., Herr, A.,    Moon, N. S., Kwon, E. J., Haigis, K. M., Naar, A. M., and    Dyson, N. J. (2008). E2F1 represses beta-catenin transcription and    is antagonized by both pRB and CDK8. Nature 455, 552-556.-   Sato, S., Tomomori-Sato, C., Parmely, T. J., Florens, L., Zybailov,    B., Swanson, S. K., Banks, C. A., Jin, J., Cai, Y., Washburn, M. P.,    Conaway, J. W., and Conaway, R. C. (2004). A set of consensus    mammalian mediator subunits identified by multidimensional protein    identification technology. Mol. Cell. 14, 685-691.-   Tsutsui, T., Umemura, H., Tanaka, A., Mizuki, F., Hirose, Y., and    Ohkuma, Y. (2008). Human mediator kinase subunit CDK11 plays a    negative role in viral activator VP16-dependent transcriptional    regulation. Genes Cells 13, 817-826.-   Westerling, T., Kuuluvainen, E., and Makela, T. P. (2007). Cdk8 is    essential for preimplantation mouse development. Mol. Cell. Biol.    27, 6177-6182.

What is claimed is:
 1. A compound represented by the structural formula:

wherein R⁶ is CN, wherein R¹³ is selected from the group consisting of2-, 3-, or 4-H, F, Cl, Br, I, OH, OCH₃, CH₃, CF₃, NR¹¹R¹², CH₂R¹¹R¹²,CO₂H and CONR¹¹R¹²; and each R¹¹ and R¹² is independently H or C1-C6alkyl, or R¹¹ and R¹² taken together form a ring.
 2. The compoundaccording to claim 1, wherein R¹³ is CONR¹¹R¹².
 3. The compoundaccording to claim 1, wherein R¹¹ and R¹² taken together form a ring. 4.The compound according to claim 1, wherein R¹³ is selected from thegroup consisting of 2-, 3-, or 4-H and Cl.
 5. The compound according toclaim 1, wherein R¹³ is Cl.
 6. The compound according to claim 1,wherein R¹³ is H.